Anal. Chem. 1998, 70, 328-331
High-Affinity RNA as a Recognition Element in a Biosensor F. Kleinjung,† S. Klussmann,‡,§ V. A. Erdmann,‡ F. W. Scheller,† J. P. Fu 1 rste,‡,§ and F. F. Bier*,†
Institute of Biochemistry and Molecular Physiology, University of Potsdam c/o MDC Max-Delbru¨ ck-Centre, Robert-Ro¨ ssle-Strasse 10, D-13125 Berlin, Germany, and Institute of Biochemistry, Freie Universita¨ t Berlin, Thielallee 63, 14195 Berlin, Germany
A biosensor for L-adenosine employing high-affinity RNA as binder is reported. Real-time measurement is obtained using total internal reflection fluorescence in a fiber-optic format. High-affinity RNA was attached to the core of a multimode fiber via an avidin-biotin bridge. The sensor measures binding of FITC-labeled L-adenosine. The interaction was fully characterized by the determination of association and dissociation rates: kD ) 0.0119 s-1; kA ) 2200 M-1 s-1. Competitive inhibition with Ladenosine enables this device to detect L-adenosine in the submicromolar range. Chemo- and biosensors challenge instrumental analytics by ease of use, sparing time, and expenditure. The analytical power of such devices derives from the molecular recognition element that is linked to a physical transducer. Molecular recognition elements are either catalysts or affinity binders. Catalysts transform the analyte, and the sensor gains a signal by measurement of the catalytic activity. Affinity binders use the binding event to extrude a signal correlated to the amount of the bound analyte. Several binders have been investigated for analytical power including antibodies, receptors, and synthetic molecular imprints.1 The antibody-antigen binding in immunosensors has become paradigmatic for sensors of the affinity type. Nevertheless, there are problems of stability and regenerability that are still unsolved. Moreover, the antibodies cannot be raised against all analytes of interest. The immunosystem usually prevents the production of antibodies against constituents of the body, such as amino acids and nucleotides. In addition, there are limitations to the production of antibodies against toxic substances for the host animal. The future development of new biosensors is likely to benefit from recent advancements in the understanding of the basic nature of nucleic acids. This understanding has come about with the rejection of the idea that nucleic acids are mere strings of sequence and their realization as molecules that fold into defined three-dimensional structures.2 The power of this method derives from the inherent nature of some nucleic acids to combine both, a genotype (nucleotide sequence) and a phenotype (ligand binding †
University of Potsdam. Freie Universita¨t Berlin. § Present address: Noxon Pharma AG, Gustav-Meyer-Allee 25, 13355 Berlin, Germany. (1) For recent review, see e.g.: Scheller, F. W., Schubert, F., Fedrowitz, J. Eds. Frontiers in Biosensors; Birkha¨user Verlag: New York, 1997. ‡
328 Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
or catalytic activity), into one molecule. Taking advantage of this unique property, oligonucleotide ligands with high affinities for a striking variety of molecular targets have been identified through directed evolution. Starting from a combinatorial library, RNA ligands are iteratively selected by affinity and amplified. This procedure for identifying oligonucleotide ligands is called systematic evolution of ligands by exponential enrichment (SELEX3). Recently, the notion of using such binders in affinity analysis has been explained.4 Here we report the use of a high-affinity RNA as a molecular recognition element in a biosensor. An L-adenosine-specific RNA5 was immobilized via a biotin-avidin bridge to an optical fiber core. Binding and competition was measured using total internal reflection fluorescence of L-adenosine conjugated to fluorescein isothiocyanate. EXPERIMENTAL SECTION Chemicals. Avidin was from Sigma (Deisenhofen, Germany). Glutaraldehyde and fluorescein isothiocyanate (FITC) were from Fluka (Neu-Ulm, Germany). The rinsing buffer included the following: 40 mM Tris-HCl, 0.25 M NaCl, pH 7.4, 5 mM MgCl, 0.1 mM EDTA. The fibers were multimodal hard clad silica (HCS) fibers of 400 µm diameter from Ensign-Bickford Optics Co. (Simsbury, CT). Chemical Synthesis of Biotinylated Oligoribonucleotides. Solid-phase synthesis of the 58-mer RNA (5′-CUC′GGU′ACC′GCA′AAA′GCG′UUU′UUC′GCA′UAC′CUA′UUC′GUU′AUA′GGU′CGA′UUG′UAC′CGA′G-3′-biotin) was performed on a PCR-MATE EP Model 391 DNA synthesizer (Applied Biosystems) essentially as described.6,7 The column material was loaded with biotin (BiotinTEG-CPG 500, Glen Research), resulting in a biotin modification at the 3′-end of the RNA. To maintain high coupling efficiencies of our 2′-O-triisopropylsilyl-protected phosphoramidites, we dissolved the amidites in acetonitrile to a concentration of 0.15 M and extended the coupling step to 900 s. The deprotection procedure was performed as described.8 After complete deprotection, the crude material was purified on 10% (2) Gold, L.; Polisky, B.; Uhlenbeck, O.; Yarus, M. Annu. Rev. Biochem. 1995, 64, 763-797. (3) Tuerk, C.; Gold, L. Science 1990, 249, 505-510. (4) McGown, L. B.; Joseph, M. J.; Pitner, J. B.; Vonk, G. P.; Linn, P. Anal. Chem. 1995, 67, 663A-668A. (5) Klussmann, S.; Nolte, A.; Bald, R.; Erdmann, V. A.; Fu ¨ rste, J. P. Nat. Biotechnol. 1996, 14, 1112-1115. (6) Scaringe, S. A.; Francklyn, C.; Usman, N. Nucleic Acids Res. 1990, 18, 54335441. S0003-2700(97)00648-3 CCC: $15.00
© 1998 American Chemical Society Published on Web 01/15/1998
Figure 1. Scheme of fiber-optic device. A flow-through cell with an embedded uncladded fiber end is connected to a sampler and samples are drawn by a peristaltic pump. The excitation light is passed through an interference filter and is guided by a fiber bundle to the measuring cell. The sensing fiber guides the fluorescence light through a long-pass filter to a photomultiplier tube. The PMT signal is collected by a lock-in amplifier which was triggered by the flash of the Xe lamp using a photodiode. All parts are connected to a PC to coordinate the flow regime and data sampling.
denaturing polyacrylamide gels (7 M urea). The band containing the product was visualized by UV shadowing and excised. The biotinylated 58-mer was eluted with water from the gel slices. The oligoribonucleotide was purified using Sephadex G-25 columns (Pharmacia), evaporated to dryness, and resuspended in water. The RNA concentration was determined by UV spectroscopy. L-AdenosineFITC Synthesis. N6-[(6-Aminohexyl)carbamoylmethyl]-L-adenosine was synthesized from L-adenosine5 as described.9 FITC (isomer I, Fluka, 41.5 mM in acetone) was added to N6-[(6-aminohexyl)carbamoylmethyl]-L-adenosine (10.5 mM in 50 mM sodium phosphate, pH 8.0) to achieve final concentrations of 8 mM nucleoside and 10 mM dye. The mixture was left for 2 h at room temperature. The product L-adenosineFITC was purified by RP-HPLC (Beckman ODS, 5 m, 80 Å, 250 mm (4 mm)) applying a gradient of 1-70% buffer B (buffer A, 100 mM triethylammonium acetate, pH 7.0; buffer B, 80% acetonitrile in A) in 30 min at 45 °C and a flow rate of 1 mL/min for 30 min. The product eluted at 20.1 min. The concentration of FITC bound to N6 -[(6-aminohexyl)carbamoylmethyl]-L-adenosine was determined by optical absorbance measurements using the molecular extinction coefficient 494 nm ) 76 000 L mol-1 cm-1.9 Fiber-Optic Fluorescence Sensor. Apparatus. The fiberoptic apparatus is schematically presented in Figure 1 and has been described previously.10,11 It consisted of a Xe flash (Hamamatsu) for excitation. The light was passed through an interference filter (480 nm, band width
